Myocardial ischemia is a major cause of hospitalization, morbidity and mortality worldwide. The phase of reperfusion, although essential for restoring blood supply to the ischemic heart, also leads to a paradoxical suppression of the myocardial function. This phenomenon is called reperfusion injury. The mechanisms of reperfusion injury have been extensively studied, and there is good evidence for a key role of free radical production and related cytotoxicity in various experimental models of reperfusion injury. We and others have obtained in vitro and in vivo evidence for the importance of a potent oxidant, peroxynitrite (formed through the rapid reaction of NO and superoxide), which initiates a number of cytotoxic reactions in the reperfusion phase. We have also obtained evidence in vitro in cardiac myoblasts, endothelial cells, and vascular smooth muscle cells, that peroxynitrite, and hydrogen peroxide, potent oxidants produced in myocardial reperfusion injury, trigger a cascade culminating in cellular energetic failure and cell death (necrosis and apoptosis). The intracellular mechanisms are multiple (and act in parallel or in synergy). Known pathways of peroxynitrite- mediated cell death involve tyrosine nitration, protein oxidation, antioxidant depletion, DNA injury, and activation of the nuclear enzyme poly (ADP- ribose) synthetase (PARS). Agents that selectively catalyze the decomposition of peroxynitrite may be novel therapeutic agents for myocardial reperfusion injury. The applicants are developing novel classes of potent peroxynitrite decomposition catalysts, which have substantial cardioprotective potential. In the current application, we first propose to test the efficacy of novel peroxynitrite decomposition catalysts in vitro. Selection of the best compounds in the in vitro assays will be followed up by studies in rat and rabbit models of myocardial ischemia-reperfusion injury. In addition, in-house preclinical safety and toxicity studies are proposed with selected decomposition catalysts. Inotek's long-term intent is to develop a selected peroxynitrite decomposition catalyst as a drug candidate for the treatment of reperfusion injury. Based on the results of the present application, Inotek will submit application for Phase 2 SBIR funding to support: pre-clinical pharmaceutical testing (advanced and formal toxicity determinations, pathology, stability, pharmacokinetics, in vivo efficacy), investigational drug application to the FDA, and a Phase 1 clinical trial. PROPOSED COMMERCIAL APPLICATIONS: The annual anticipated revenue for an effective therapeutic to prevent and treat myocardial repertusion injury is estimated at> $1 billion in the US per annum.